3-cyclohexene-1-glyoxylic acid, salts and esters



United States Patent 3,271,439 3-CYCLOHEXENE-l-GLYOXYLIC ACID, SALTS ANDESTERS Walter D. Celmer, New London, Conn, assignor to Chas. Pfizer &Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. FiledMar. 5, 1963, Ser. No. 262,829 5 Claims. (Cl. 260468) This inventionrelates to organic compounds which are useful as antibiotic substances.More particularly, it is concerned with 3-cyclohexene-l-glyoxylic acid,with alkali metal and alkaline metal salts thereof and with its esterderivatives. The invention also contemplates processes, bothfermentative and synthetic, for the prep aration of the valuable newglyoxylates.

This invention relates specifically to compounds of the formula:

wherein R is selected from the group consisting of hydrogen, alkalimetals, alkaline earth metals and alkyl groups containing from 1 to 3carbon atoms.

It will be recognized that the ring carbon atom to which the glyoxylicgroup is attached is asymmetric, that is, it can be said to have fourdifferent radicals or atoms attached to it. By virtue of this, thecompounds contemplated by the instant invention exist either asoptically active forms, the so-called stereoisomeric3-cyclohexenel-glyoxylates, or as mixtures thereof. It has now beenfound that those optically-active stereoisomers of the compounds of theinstant invention which turn the plane of polarized light to the right,that is, clockwise and which are hereinafter referred to as thedextrorotatory forms, or the (+)-forms, possess antibiotic activity.This antibiotic activity is particularly pronounced against Gramnegativeorganisms and is accompanied by a substantial lack of acute toxicmanifestations.

Particularly effective as an antibiotic is dextrorotatory(+)-3-cyclohexene l-glyoxylic acid, the compound of the formula abovewherein R is hydrogen. This useful activity against a variety oforganisms permits the application of the microbiologically-activestereoisomeric forms of the instant compounds in therapeutics, inveterinary medicine and in agriculture. The new antibiotic compounds arealso of use in industrial fermentations to prevent contamination bysensitive organisms. They have activity in medical diagnostic techniquesand, in addition, are useful in separating and classifying organisms.

With respect to the counterparts of the dextrorotatory compounds of theinstant invention, those which, in contrast, turn the phase of polarizedlight to the left, that is, counterclockwise and which are hereinafterreferred to as the levorotatory forms, or the ()-forms, these are usefulas intermediates in the preparation of the microbiologically-activedextrorotatory forms. Thus the levorotatory form may be racemized, thatis, converted by physical or by chemical means, to a mixture of the(-1-)- and ()-forms, conveniently, for example, by being treated with abasic reagent such as potassium hydroxide, and then the active (+)-formmay be recovered from the mixture by the commonly employed techniqueknown as resolution. Resolution contemplates, in essence, the separationof a racemic mixture into its optically-active components. Suchseparations may be effected by, for example, fractional crystallizationswhich are based on differences in solubility between the stereoisomersor derivatives thereof.

3,271,439 Patented Sept. 6, 1966 The compoundD,L-3-cyclohexene-l-glyoxylic acid may be readily obtained by synthesis.For example, the following outlined route is especially convenient:

The starting material is the well-known and readilyavailable adduct ofbutadiene and acrolein. This is caused to react with ammonium carbonateand sodium cyanide, Step (1), and the intermediate formed thereby istreated with barium hydroxide and Water, Step (2), to form3-cyclohexene-1-glycine, substantially in accordance with the procedureset out by Edelson et al., in volume 80, Journal of the AmericanChemical Society, pages 2698 et seq, 1958.

With respect to step (3), 3-cyclohexene-1-glycine is converted to3-cyclohexene-l-glyoxylic acid by treatment with pyridoxalmonohydrochloride. It has been found to be particularly convenient touse a modification of the method of Metzler and Snell, volume 74,Journal of the American Chemical Society, page 979, 1952, wherein aphosphate buffer is substituted for the acetate buffer described thereinfor similar transamination reactions. For example, pyridoxalmonohydrochloride, dissolved in about 500 parts of water, is treatedwith an equivalent amount of 3-cyclohexene-l-glycine and equivalent ofaluminum ammonium sulfate dodecahydrate per 10 equivalents of pyridoxaloriginally taken, is added. The solution is stirred at about 25 C. forabout 15 minutes, then the pH, which is about 3.3, is increased to about5.0 by the addition of an appropriate amount of 0.75 ammonium hydroxidesolution. The resulting mixture is stirred and is heated to about C.during about 1 hour. The mixture is cooled to about 25 C. and is treatedwith enough 10% aqueous phosphoric acid to lower the pH to about 2.5,then is extracted with an equal volume of methylene chloride. Theaqueous layer is adjusted to pH 5.0 by the addition of an appropriateamount of 5 N sodium hydroxide solution, is concentrated in vacuo toabout /3 volume and then is refluxed for 16 hours. The reaction mixtureis cooled to about 25 C. and is extracted with an equal volume ofmethylene chloride. The aqueous layer is adjusted to about pH 2.0 withan appropriate quantity of 50% phosphoric acid and the solution isextracted with an equal volume of methylene chloride. The organic layeris separated, is dried with anhydrous sodium sulfate, the drying agentis removed by filtration and the solvent is removed by distillation. Theproduct, racemic 3 cyclohexene'l-glyoxylic acid remains as a white,crystalline residue, M.P. 45-50 C. The product can be further purifiedby recrystallization from heptane, from which it is deposited asrectangular plates, M.P. 5556 C.

In addition to the hereinbefore described synthetic process, thedextrorotatory form of 3-c-yclohexene-lglyoxylic acid may be obtainedby'a fermentation process. Thus, the present invention contemplates aprocess for the preparation of (+)-3-cyclohexene-1-g1yoxylic acid whichcomprises cultivating Streptomyces antibiotic-us, strain ATCC 14890, inan aqueous nutrient medium containing a carbohydrate and a source oforganic nitrogen and inorganic salts under submerged aerobic conditionsuntil substantial anti-microbial activity is imparted to said 3 mediumand recovering said acid from the fermentation broth. 7 1

The organism which is employed in the valuable fermentation process ofthe present invention has been identified to be a new strain of aspecies of microorganism known as Streptomyces antibioticus.Identification was made by planting and testing a culture of theorganism on media normally used for such identifications and the mediawere incubated at controlled temperatures for 14 days. Readings weremade at intervals and final observations were recorded after two weeksof incubation. A culture of this strain has been deposited in theAmerican Type Culture Collection, Washington, DC, and has been assignedthe number ATCC 14890.

The description of this microorganism, designated FD 13219 in theculture collection of Chas. Pfizer & Co., Inc. of Groton, Connecticut,was supplied by Dr. J. B. Routien. The cultural characteristics observedand recorded for this strain are set forth in Table I ((R) refers toRidgways Color Standards and Nomenclature):

genetic recombination or some other genetical procedure using a nucleicacid or an equivalent material from the herein described species wherebyit has acquired the ability to produce the elaboration product describedherein and claimed.

Strain ATCC 14890 will grow on many media used for the propagation ofStreptomycetes. A satisfactory medium is Pridhams Yeast Extract Agar: Amixture of 4.0 g. of yeast extract, 10.0 g. of malt extract and 4.0 g.of dextrose in 1000 ml. of water is adjusted to pH 7.3 with sodiumhydroxide, 20.0 g. of agar is added, the mixture is steamed for 15 tominutes, dispensed, and then is sterilized for 15 minutes at 121 degreesC. The culture should be incubated at 28 degrees C.

(+)-3-cyclohexene-l-glyoxylic acid and its alkali metal and alkalineearth metal salts and its ester derivatives are remarkably effective intreating a number of infections. They exhibit significant activityagainst a wide variety of microorganisms. However, they are particularlynoteworthy in their action on Gram-negative or- TABLE I.CULTURALCHARACTERISTICS OF FD-13219 Medium Amount of Growth Aerial Mycelium andSpores Soluble Pigment Remarks Glucose asparagine agar. Good Good,nearLight Mouse Light Brown Reverse pale yellow at edge Gray (R). andbecoming brown in center. Synthetic agar Very poor Very thin, palecinnamon. Lacking Reverse colorless. Gauzesinorganic medium Good. Good,Light Mouse Gray Gray Brown Reverse brown; chains of N0. 1. to MouseGray (R). spores straight to flexuous and in loops, scattered alonghyphae', spores broadly elilptical, 1.3-24) X 0.7-1.0 G. Calcium lnalatcagar Poor, flat Poor, nearMouse Gray Lacking Reverse light yellow;

(B). calcium malate weakly digested. Cellulose Good. Abundant,nearuNlouse .--..do

skimmed milk Potato plug Nutrient agar.. Glucose agar... Emerson's agarPridhams yeast extract agar. Hickey dz 'Iresners agar. Gelatin Starchagar Peptone iron agar Moderate in brown ring.

Good Peer to moderate..

Excellent Good Moderate, flat Poor, fiat G ray (R). Lacking Good, grayPoor, grayish white. Good, grayish white Good, light grayish white...

Moderate, near Olive Gray to Mouse Gray Dark brownin upper hydrolyzedportion; in lower part near Ridgways Sayal Brown.

Gray to black Pale yellowish brown Dark brown No coagulation; hydrolysispartial; pH change from 6.5 to 6.9.

Vegetative rnycelium yellow Reverse whitish.

Reverse grayish tan.

Reverse grayish tan, vegetative inycelium yellow.

Reverse yellow.

' like.

In connection with the description of this organism, it has beenobserved occasionally that there is a tendency for the colors impartedto some media to vary somewhat. This is especially pronounced after anumber of transfers have occurred and the reasons for this are notclearly understood at this time. However, this variation in appearancedoes not have a marked effect on the ability of the organisms to producethe antibiotic substance of the present invention.

It is to be understood that for the production of3-cyclohexene-l-glyoxylic acid, the present invention is not limited tothis particular strain or to a strain fully answering the abovedescription, which is given only for illustrative purposes. It isespecially desired and intended to include the use of mutants producedfrom the described organism by various means such as X-radiation,ultraviolet radiation, nitrogen mustards and the It is contemplated alsoto include any organism regardless of its appearance or physiologicalbehavior,

that may be developed by transformation, transduction,

ganisms. While they demonstrate some activity against Gram-positiveorganisms, this activity is generally of a somewhat lower level.

The invitr-o activity of (+)-3-cyclohexene-1-glyoxylic acid against agroup of microorganisms which cause various diseases is demonstratedagainst strains of Escherichia coll, Proteus vulgaris, A. aerogenes andSalmonella typhosa. In vivo, it demonstrates the ability to protect miceagainst death after being infected with E. coli; for example,(-1-)-3-cyclohexene-1-glyoxylic acid protected 50% (five out of ten, PDmice infected IP with E. coli strain 462 after a multiple oral dosage/2, 4, 24, 48 hours post infection) of 800 rng/k After administration bythe subcutaneous route, the corresponding PD was found to be 500 mg./kg.The potassium salt of (+)-3-cyclohexene-1-glyoxylic acid exhibits lowacute toxicity in rats; no deaths are observed after dosages of 2000mg./kg. orally or 300 rug/kg. intravenously. The

intravenous LD was 400 mg./kg.

It is to be understood that for the purpose of treatmerit of infectionswith the valuable compounds of the instant invention, either the purematerials or one of the crude forms of the antibiotic may be used. Thisis to include either a filtered fermentation broth, as produced from,for example, strain ATCC 14,890, or a solid or liquid concentrateprepared therefrom. For administration to man and animals, a non-toxiccarrier is, of course, selected, toxicity for this purpse being definedas an adverse effect on the treated host at the level of ordinary use.Either liquid or solid pharmaceutical carriers may be employed,including water, aqueous ethanol, isotonic saline or glucose, starch,lactose, calcium phosphate, animal feed stuffs, or mixtures of variousmaterials as occur in a filtered fermentation broth. Either oral orparenteral administration is satisfactory, although the parenteral routeis perhaps preferable until a satisfactory regimen adapted to thepatient is established. For this purpose, solutions of suspensions inwater, oils, such as peanut oil, or other pharmaceutically acceptablesolvents or vehicles may be employed. Solid preparations forextemporaneous dilution can be prepared containing various bufferingagents, local anaesthetics, and other medicinal agents includingantibiotics, hypnotics, analgesics, as well as inorganic salts to afforddesirable pharmaceutical properties to the composition.

The present invention embraces the process for growing Streptomycesantibioticus, ATCC 14890, under controlled conditions to produce(+)-3-cyclohexene-1-glyoxylic acid. The culture is grown submerged in anaqueous nutrient medium containing a source of nitrogen, a carbohydrateand minerals.

A satisfactory medium contains 10 g./l. of Cerlose (dextrose hydrate),g./l. of Curbay-BG (molasses residue), g./l. of cornstarch, 10 g./l. ofsoybean meal, and 5 g./l. of sodium chloride. The medium is adjusted topH 7.0 with 1 N potassium hydroxide, calcium carbonate, 10 g./l., isadded and broth is sterilized at 121 C. for minutes. It is inoculatedwith 27% of a -60 hour old culture grown in the same medium. Per- 8tate, methyl isobutyl ketone and methylene chloride, respectively.

The crystalline antibiotic may be further purified by recrystallizationof its potassium salt from a mixture of methanol and isopropanol. Theless pure acid is dissolved in about 10 parts by Weight of methanol togive a 1.34.4 molar acid solution. Then 4 N methanolic potassiumhydroxide, in an amount equivalent to the acid originally taken, isadded and the precipitated solid is removed by filtration. The filtrateis concentrated to about one-half volume and 1.5-2.0 volumes ofisopropanol is added, with stirring. The potassium salt which isprecipitated is removed by filtration and is dried. A second crop isobtained by concentration of the mother liquor.

It has been surprisingly found that the crystalline antibiotic of theinstant invention is also produced by the strain ATCC 11891 ofStreptomyces antibioticus, which strain was taught in US. 2,757,123, asproducing the basic antibiotic, oleandomycin, described and claimedtherein; the strain disclosed in the said patent is being maintained, onpublic deposit, in the American Type Culture Collection, Washington, DC.It is now found that strain ATCC 11891, in addition to producingoleandomycin, also produces the crystalline acidic antibiotic of theinstant invention. In contrast, the first mentioned strain of theinstant application, ATCC 14890, produces only(+)-3-cyclohexene-1-glyoxylic acid and no oleandomycin. Co-production ofthe new antibiotic by the old strain ATCC 11891 has not been observedbefore, since the strain has been used to produce a basic antibiotic andthe acidic antibiotic of the instant invention has been discarded in thesewer liquor without having been characterized or isolated.

In addition to the inability for strain ATCC 14890 to produceoleandomycin, there are other significant differences in growth behaviorbetween it and the oleandornycin-producing S. antibioticus ATCC 11891described in the above-mentioned patent; these differences aresummarized in Table II:

DIFFERENCES BETWEEN ATCC 11891 AND ATCC 14890 Medium I ATCC 11891 ATCC142390 Potato plu Emersous Stare-IL." Calcium inalate agar Gauzesinorganic medium No.

Vegetative inycelium yellow. Colonies with grayish white spores. Norings of growth.

Weak digestion of malate.

Soluble pigment dark brown. Reverse brown.

mentation can be carried out at about 26-30 C. in 4 liter stirred glassjars, mechanically aerated, for from about to about 130 hours. Theprogress of the fermentation can be followed by standard plate assaymethods using the activity of the broth against Aerobacter aerogenes.

After a satisfactory level of antibiotic activity has been obtained, theactive substances may be isolated by procedures well known to thoseskilled in the art. A particularly satisfactory procedure is outlined asfollows: The broth is filtered and then is adjusted to a pH of 25:1 andis extracted with one-third to one-half volume of methylisobutyl ketoneor ethyl acetate. The organic layer is partitioned against aqueoussodium phosphate solution, the aqueous layer is separated and isextracted with an equal volume of methylene chloride. The methylenechloride layer is separated and is treated with an equal volume ofcyclohexene or heptane and concentration of the resulting solution toabout one-fourth volume causes the precipitation of crystalline(+)-3-cyclohexenel-glyoxylic acid. The term substantiallywater-immiscible organic solvent as used in the appended claimscontemplates those dissolving )-3-cyclohexene-1-glyoxylic acid and beingof the type indicated, for example, esters, ketones and chlorinatedhydrocarbons such as ethyl ace- In order to recover the valuable newacidic antibiotic from broths produced by the fermentation of strainATCC 11891, the sewer liquors, which have up until now been discardedafter removing oleandomycin, are subjected to the following procedure:The sewer liquor broth is adjusted to a pH of from about 1 to about 5 bythe addition of phosphoric acid. About A; volume of methylisobutylketone is added and stirring is continued for about 15 minutes, then theaqueous layer is drawn off and is discarded. The organic layer isextracted with 6 one-fourth volume portions of 0.35% disodium phosphatesolution and the organic layer is discarded. The aqueous layer isextracted with about onetenth volume of methylene chloride, the organiclayer is separated and the aqueous layer is adjusted to pH 2 withphosphoric acid. The aqueous layer is then extracted with about onetenth volume of methylene chloride and the methylene chloride solutionis distilled in vacuo until free of solvent. The residue is thendissolved in about 50 parts by weight of methanol, and the solution isadjusted to pH 6710.1 with 2 N methanolic potassium hydroxide, then thesolvents are removed by distillation in vacuo. The crystalline residueis triturated with isopropanol, the crystals are removed by filtrationand are dried.

As has been mentioned hereinbefore, advantage is taken of theinterconvertibility of optically-active forms in the synthesis of themicrobiologically active (+)-3- cyclohexene-l-glyoxylic acid and itsderivatives. Thus the microbiologically inactive forms are racemized andthe desired isomer is isolated by resolution of the mixture. Thestereoisomers, though very stable at neutral and acidic pHs, areracemized with increasing rapidity as the alkalinity of the solutionincreases. For example, at the following pH values, there remains therespective percentages of original rotation after 19 hours: pH 7, 99%;pH 8.9, 86%; pH 9.5, 78%; pH 10.4, 45%; and pH 11.5, At pH 12.0, alloptical activity is lost in about 30 minutes; all measurements were madeon the potassium salt in buffered phosphate aqueous solutions at C.,except for the unbuffered measurement at pH 12.

For purposes of preparing the (+)-form from the ()-form, it isespecially convenient to racemize the potassium salt in sodium hydroxidesolution and to separate the mixture of and ()-forms, which results, byfractional crystallization of salts with optically active basicalkaloids by techniques well known in the art. Thus, the potassium saltof the optically-active form is dissolved in about parts of water andthe solution is rendered basic to pH 12.2 by addition of ION aqueoussodium hydroxide. Progress of the racemization is readily followed in apolarimeter and, after about 1.5 hours at 25 C., the solution has lostits rotation. After an additional half-hour the racemate may be isolatedby adjusting the solution to pH of about 2 with 50% phosphoric acid,extracting with an equal volume of methylene chloride, separating theorganic layer, drying the organic layer and evaporating the solventtherefrom. The racemic product remains as a crystalline residue whichcan be separated into pure and ()-forms by successive slowcrystallizations of salts with optically active basic alkaloids, themother liquor being removed for further crystallization followed byregeneration of the optically active acids.

Of course, in addition to the synthesis of (+)()-3-cyclohexene-l-glyoxylic acid from 3-cyclohexene-1-glycine, it will beobvious to those skilled in the art that alternative methods areavailable for its preparation. Among these are, for example:

The reaction of butadiene and certain selected acids in accordance withthe following sequences *Instead of this compound, the Mannich Base,

ReNCHzCHzC O) C0211 or Mannich Base N-oxide, R2N( O) CHaCHzC (O) COaH,may be employed,

The following examples are given by way of illustration and are not tobe construed as limitations of this invention, many variations of whichare possible without departing from the spirit or scope thereof.

Example I A solution of 1018 mg., 5 meq., of pyridoxal monohydrochloride(Nutritional Biochemical Corporation, Cleveland, Ohio) in 500 ml. ofwater is treated with 3-cyclohexene-l-glycine, 775 mg, 5 meq., preparedby the process of Edelson et al., volume 80, ].A.C.S., page 698, 1958(substituting a phosphate buffer for the corresponding acetate buffer),and 227 mg., 0.5 meq., of aluminum ammonium sulfate dodecahydrate. Theresulting mixture is stirred at 25 C. for 15 minutes, then the pH isadjusted from an initial 3.3 to a final 5.0 by the addition of 6.6 ml.of 0.75 N ammonium hydroxide solution. The mixture is then heated to C.during 60 minutes, with stirring. The mixture is cooled to about 25 C.,then is adjusted to a pH of 2.5 by the addition of 10 ml. of 10%phosphoric acid. The acidic mixture is extracted with 500 ml. ofmethylene chloride and the organic layer is separated. The aqueous phaseis adjusted to pH 5.0 with 5 N sodium hydroxide, is evaporated to aboutone-half volume in vacuo to remove residual methylene chloride, then isrefluxed for 16 hours. The resulting solution is cooled to 25 C. and isextracted with an equal volume of methylene chloride; the methylenechloride layer is separated and is discarded. The aqueous phase isadjusted to pH 2.0 with 50% phosphoric acid and is extracted with anequal volume of methylene chloride. The solvent layer is collected, isdried with sodium sulfate, the sodium sulfate is removed by filtrationand the solvent is distilled leaving the product,3-cyclohexene'l-glyoxylic acid, as a white, crystalline residue, 210mg., M.P. 4550 C. The product is recrystallized from heptane and isdeposited as rectangular plates, M.P. 55-56 C. D,L-3-cyclohexene-1-glyoxylic acid is converted to its potassium salt by dissolving the acidin methanol to afford a 1.3 molar concentration. Then is added 4 Nmethanolic potassium hydroxide until the pH is 7. A small amount ofprecipitated solid is removed by filtration, then the filtrate isconcentrated to one-half volume and 1.5 volumes of isopropanol is addedthereto. The potassium salt which precipitates is removed by filtrationand is dried.

Example II A slant of S. anribioticus ATCC 14890 on Emerson agar iscultivated to develop spores for the purpose of inoculating a nutrientmedium of the following composition:

' Grams Cerelose (dextrose hydrate) 10 Curbay-BG (molassess residue) 5Cornstarch 10 Soybean meal 10 Sodium chloride 5 This mixture ofnutrients is diluted to a volume of one liter with water, is adjusted toa pH of 7 with potassium hydroxide and is sterilized at 121 C. forthirty minutes. The medium is then cooled and the spores are addedaseptically. The organism is cultivated in Fernbach flasks on areciprocating shaker at 28 C. for two days. The mixture of broth andmycelium formed thereby is transferred to 20 volumes of a sterilefermentation medium having the same composition, adjusted to pH 7. Afterseeding the medium with the organisms from the Fernbach flasks, themixture is agitated and aerated at 28 C. for 108 hours. The potency ofthe broth is measured against A. aerogenes. The mycelium is removed byfiltration and the filtrate is adjusted to pH 2 with phosphoric acid.The acidic solution is extracted with one-fourth volume ofiethylisobutyl ketone and the separated organic layer is extracted fourtimes with equal-volume portions of a 0.35% di-sodium phosphate buffersolution. The buffer extracts are separated, are combined and are washedwith one-twelfth volume of methylene chloride; the methylene chloridelayer is separated and is discarded. The Washed bufier is adjusted to pH2 with phosphoric acid and is extracted with an equal volume of freshmethylene chlo ride, then the methylene chloride is separated and isconcentrated to dryness. The crystalline residue is triturated withcyclohexene and the crystalline (+)-3-cyclohexene-lglyoxylic acid isremoved by filtration and is dried. The crystalline product, M.P. 5556C. [alpha] =|60i5 (in Water), is found to be highly effective against avariety of gram-negative microorganisms, both in vivo and in vitro as isindicated in the description hereinbefore.

The potassium salt of the antibiotic [alpha] --+50i-5 (in water), isprepared by the procedure described in Example I.

Example [11 The procedure of Example II is repeated, substituting forstrain ATCC 14890, strain ATCC 11891. After the(+)-3-cyclohexene-l-glyoxylic acid is removed by extraction at acidicpH, the aqueous phase is adjusted to a pH of about 9 and is extractedwith chloroform. The chloroform extract is concentrated to a sirupyconsistency and, after standing, crystalline oleandomycin chloroformsolvate (cf. Celmer et al., Antibiotics Annual, 1957-1958, p. 478)formed. A methanol solution of the chloroform solvate is distilled toremove chloroform azeotropically and the halogen-free concentrate isthen cautiously treated with water until crystallization of the freeoleandomycin base is induced. This is removed by filtration and isidentical to the product of US. 2,757,123. This antibiotic in contrastto the crystalline (+)-3-cyclohexene-1-glyoxylic acid is highlyeffective against gram-positive microorganisms, as is indicated in thecited patent.

Example IV Salts of 3-cycl0lzexene-I-gly0xylic acid.-A stirred solutionof crystalline (+)-3-cyclohexene1glyoxylic acid, 1.0 g., in isopropanol,20 ml., is treated dropwise with 1 N methanolic sodium hydroxide, 6 ml.The crystalline sodium salt which forms is recovered by filtration andis dried, yield, 560 mg. In a similar manner, the sodium salt ofD,L-3-cyclohexene-l-glyoxylic acid of Example 1 is prepared.

A stirred solution of l g. of crystalline potassium (4-)-3-cyclohexene-l-glyoxylate in 10 ml. of water is treated with asaturated aqueous solution of calcium acetate, 2 ml. The crystallinecalcium salt which forms is removed by filtration and is dried, yield,660 mg. This procedure is repeated With D,L-3-cyclohexene-1-glyoxylicacid and the corresponding racemic acid salt is formed. The procedure isrepeated with stoichiometrically equivalent amounts of magnesium acetateand barium acetate; the corresponding magnesium and barium salts of(+)-3-cyclohexene-1 glyoxylic acid are obtained.

Example V Esters of 3-cycl0lrexene-Iglyoxylic acirL-Crystalline(+)-3-cyclohexene-1-glyoxylic acid, 40 g., is dissolved in ml. ofmethylene chloride and is treated With 40 ml. of methanol and 0.8 ml. ofconcentrated sulfuric acid. The solution is refluxed for 2 hrs., iscooled and filtered and the solvents are removed by distillation at 25C. at a pressure of 1515 mm. of mercury. The residue is then distilledby raising the bath temperature to about C., whereupon a first fraction,11.4 g., refractive index, 1.4735, and a second fraction, 21.3 g.,refractive index, 1.4740 are obtained at approximately 100 C. (headtemperature). By the same procedure, the methyl ester ofD,L-3-cyclohexene-1-glyoxylic acid is obtained.

The procedure is repeated substituting for methanol, equivalent volumesof ethanol and n-propanol. There are obtained, respectively, the ethyland n-propyl esters of 3- cyclohexene-l-glyoxylic acid.

What is claimed is:

1. A compound having the formula wherein R is selected from the groupconsisting of hydrogen, alkali metal, alkaline earth metal and alkyl offrom 1 to 3 carbon atoms.

2. D,L-3-cyclohexene-1-glyoxylic acid.

3. (+)-3-cyclohexene-1-glyoxylic acid.

4. Potassium (+)-3-cyclohexene-l-glyoxylate.

5. Methyl (+)-3-cyclohexene-l-glyoxylate.

References Cited by the Examiner UNITED STATES PATENTS 2,734,018 2/1956Minieri et al -180 2,771,487 11/1956 Morris et a1. 260-514 2,776,2431/1957 McGhee et al. 195-180 2,808,363 10/1957 Hoeksema et a1 195-1802,847,460 8/1958 Trapp et al. 260-514 LORRAINE A. WEINBERGER, PrimaryExaminer. A. LOUIS MONACELL, Examiner.

D. M. STEPHENS, R. K. JACKSON,

Assistant Examiners.

1. A COMPOUND HAVING THE FORMULA